玉米赤黴烯酮與脫氧雪腐鐮刀菌烯醇抗體之製備並將其應用於酵素連結免疫分析法之開發

Production of Antibodies and Development of Enzyme-Linked Immunosorbent Assay for Zearalenone and Deoxynivalenol

鐘唯恆(Wei-Heng Chung)

指導教授 ： 余豐益

中山醫學大學/醫學科技學院/生物醫學科學學系/碩士(2019年)

https://doi.org/10.6834/CSMU201900033

摘要

黴菌毒素中的玉米赤黴烯酮 (zearalenone, ZEA) 及脫氧雪腐鐮刀菌烯醇 (deoxynivalenol, DON) 皆為 Fusarium spp. 所產生的次級代謝物，由於兩者皆具有高盛行率及結構不易被破壞之特性，因此於未加工穀物或已加工食品中時常被檢測出。若人類誤食受兩者汙染之食品，可能會有頭痛、嘔吐或腹瀉等急性症狀，若長期食用則可能會有食慾不振、體重下降或肝病變等慢性症狀。因此本研究希望製備對玉米赤黴烯酮或脫氧雪腐鐮刀菌烯醇具專一性的抗體，並利用此抗體建立一套快速且靈敏的檢測方式，檢測食品中玉米赤黴烯酮及脫氧雪腐鐮刀菌烯醇的含量。由於玉米赤黴烯酮及脫氧雪腐鐮刀菌烯醇皆屬於小分子毒素，雖具有抗原性，但不具有免疫原性，因此需與載體蛋白質接合，使其具免疫原性後，對實驗動物進行免疫，以製備出對玉米赤黴烯酮或脫氧雪腐鐮刀菌烯醇具有專一性的抗體，並利用此抗體來開發酵素連結免疫吸附法 (enzyme-linked immunosorbent assay, ELISA)。於玉米赤黴烯酮部分，本研究雖尚無法藉由細胞融合瘤實驗篩選出單株抗體，但建立準確定量衍生物的方法，並成功使小鼠產生具專一性的抗體，且此抗體是足夠以檢測各國訂定最低限制含量。於脫氧雪腐鐮刀菌烯醇部分，本研究雖嘗試多種方法接合載體蛋白質並進行動物免疫，仍無具專一性的抗體產生，但本研究成功接合出兩種可用於開發 ELISA 的 DON-horseradish peroxidase (DON-HRP)。本研究的結果雖然不符合預期，但本研究所使用的方法及提出的論點，希望作為未來研究者之參考。

關鍵字

玉米赤黴烯酮 ； 脫氧雪腐鐮刀菌烯醇 ； 多株抗體 ； 酵素連結免疫分析法

並列摘要

As mycotoxins, zearalenone (ZEA) and deoxynivalenol (DON) are both secondary metabolites produced by Fusarium species. Due to invulnerability of structure and high prevalence, both ZEA and DON are frequently detected in unprocessed grains or processed food. If people consumed the contaminated food, in short term it may cause acute effects like headache, nausea, and diarrhea; in long term it may cause chronic effects like appetite loss, weight loss, and hepatic disease. Thus, producing an anti-ZEA or anti-DON antibody and developing a rapid and sensitivity detecting method based on these antibodies for ZEA and DON are the aims of present study. Both ZEA and DON belongs to small molecular weight toxins, which contain antigenicity but not immunogenicity. In order to gain immunogenicity, mycotoxins were conjugated to carrier proteins. To produce the antibody against ZEA or DON, the toxin-protein conjugates were served as an antigen to immunize the animals. The enzyme-linked immunosorbent assay (ELISA) based on the antibody describe above was developed. Despite of the failure of selecting the hybridoma cell which secreting antibody against ZEA, the present study established a method of precise measuring quantity of ZEA derivatives and successfully produced an antibody with ability detecting the lowest regulation level legislated by countries. On the other hand, to produce an antibody specific to DON, several different antigen conjugation methods and animals’ immunization had been carried out. However, none of which could efficiently produce antibody against DON, but two effective DON-horseradish peroxidase (DON-HRP) had been developed in the present study. Although the ideal isn’t fulfilled by the experimental results, it is hoped that the methods used and thesis mentioned in the present study will be fundamental to future researches.

並列關鍵字

Zearalenone ； Deoxynivalenol ； ELISA ； polyclonal antibody

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